CN112779207A - Rejuvenation method of ganoderma lucidum strain special for liquid fermentation - Google Patents
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- 240000008397 Ganoderma lucidum Species 0.000 title claims abstract description 59
- 235000001637 Ganoderma lucidum Nutrition 0.000 title claims abstract description 57
- 238000000855 fermentation Methods 0.000 title claims abstract description 39
- 230000004151 fermentation Effects 0.000 title claims abstract description 39
- 230000003716 rejuvenation Effects 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000007788 liquid Substances 0.000 title claims abstract description 20
- 238000012216 screening Methods 0.000 claims abstract description 25
- 230000004071 biological effect Effects 0.000 claims abstract description 16
- 238000012807 shake-flask culturing Methods 0.000 claims abstract description 12
- 238000012795 verification Methods 0.000 claims abstract description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 26
- 239000008103 glucose Substances 0.000 claims description 26
- 239000000758 substrate Substances 0.000 claims description 22
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 9
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 9
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 9
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- 239000001965 potato dextrose agar Substances 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 230000007850 degeneration Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 238000009630 liquid culture Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract 1
- 241000222336 Ganoderma Species 0.000 description 7
- 241000233866 Fungi Species 0.000 description 6
- 239000013543 active substance Substances 0.000 description 3
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 241000222341 Polyporaceae Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
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- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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Abstract
The invention discloses a rejuvenation method of a ganoderma lucidum strain special for liquid fermentation, which comprises the following steps: (1) shake flask culture screening: transferring the activated parent ganoderma lucidum mycelia into a shake flask for culture, and screening shake flask fermentation liquor with biological activity indexes equivalent to those of the parent ganoderma lucidum mycelia for later use; (2) plate culture screening: inoculating the fermentation liquor obtained by screening in the shake flask and equivalent to the parent biological activity index into a flat plate again for culturing, and selecting the flat plate with high growth speed for later use; (3) and (3) shake flask culture verification: transferring the cultured ganoderma lucidum mycelia on the spare plate into a shake flask for subculture verification, wherein the plate strain used by shake flask fermentation liquor with biological activity indexes equivalent to those of parents and stable passage is the rejuvenated ganoderma lucidum strain. The method can effectively solve the problems of strain degeneration, weak strain activity and poor strain quality caused in the process of passage of liquid culture of the ganoderma lucidum strain, and provides strain guarantee for liquid large-scale sustainable fermentation production of the ganoderma lucidum strain.
Description
Technical Field
The invention relates to the technical field of microbial strain breeding, in particular to a rejuvenation method of a ganoderma lucidum strain special for liquid fermentation.
Background
Ganoderma lucidum is a fungus belonging to the genus Ganoderma of the family Polyporaceae of the class Basidiomycetes, and is one of the famous and precious medicinal fungi in China. It is recorded in Shen nong Ben Cao Jing of Han Dynasty and has the functions of strengthening body resistance, consolidating constitution, nourishing, prolonging life, maintaining balance of body constitution, etc. In recent years, ganoderma lucidum has been intensively studied by many scholars both domestically and abroad. The method for obtaining the ganoderma lucidum active ingredient materials at the present stage comprises three methods, namely field sporocarp or artificially cultivated sporocarp, ganoderma lucidum spore, fermented mycelium and fermentation liquor. Compared with artificial cultivation of ganoderma, the production of ganoderma lucidum active substances by fermentation technology has the characteristics of high hypha proliferation speed, no influence by seasons, short production period, relatively stable content of active ingredients and the like, is a novel and important technical means for obtaining ganoderma lucidum active substances with stable quality, and has attracted wide attention of scholars at home and abroad.
In recent years, most researches on liquid submerged fermentation of ganoderma lucidum have focused on fermentation culture media, optimization of fermentation conditions, analysis of characteristics and components of fermentation products and the like, and attention is paid to improvement of yield of active substances such as ganoderma lucidum polysaccharide, and few researches on ganoderma lucidum strains special for liquid fermentation are performed. At present, most strains for ganoderma lucidum liquid state fermentation are naturally bred, the properties of many ganoderma lucidum strains are unstable and are easy to degenerate and age after several generations of subculture, so that the high-quality properties of the ganoderma lucidum strains are difficult to maintain for a long time.
Therefore, a rejuvenation technology of ganoderma lucidum strains special for liquid fermentation is urgently needed at present.
Disclosure of Invention
The invention aims to provide a rejuvenation method of a ganoderma lucidum strain special for liquid fermentation, which comprises the following steps:
(1) shake flask culture screening:
transferring the activated ganoderma lucidum mycelia of the parent strains into a shake flask for culture, and screening shake flask fermentation liquor with biological activity indexes equivalent to those of the parents for later use;
wherein the formula of the shake flask culture medium is as follows (unit: g/L): 10-50 parts of glucose, 2-10 parts of yeast powder, 1-8 parts of monopotassium phosphate and 1-8 parts of magnesium sulfate heptahydrate;
(2) plate culture screening:
inoculating fermentation liquor which is obtained by screening in a shake flask and is equivalent to the biological activity index of a parent into a flat plate for culture, and selecting the flat plate with high growth speed for later use;
wherein the formula of the plate culture medium is as follows (unit: g/L): 10-50 parts of glucose, 2-10 parts of yeast powder, 1-8 parts of monopotassium phosphate, 1-8 parts of magnesium sulfate heptahydrate and 10-20 parts of agar; or using potato dextrose agar (PDA medium) plates;
the growth rate refers to the time taken for the ganoderma lucidum mycelia to grow over the flat plate;
(3) and (3) shake flask culture verification:
transferring the cultured ganoderma lucidum mycelia on the spare plate into a shake flask for subculture verification, wherein the plate strain used by shake flask fermentation liquor with biological activity indexes equivalent to those of parents and stable passage is rejuvenated ganoderma lucidum strain;
wherein, the formula of the shake flask screening is as follows (unit: g/L): 10-50 parts of glucose, 2-10 parts of yeast powder, 1-8 parts of monopotassium phosphate and 1-8 parts of magnesium sulfate heptahydrate;
the stable passage means that the fluctuation range of the mycelium yield and the utilization rate of substrate glucose is within 10 percent after each passage;
wherein the biological activity indexes in the steps (1), (2) and (3) comprise the yield of mycelium and the utilization rate of substrate glucose.
Wherein the parent strain is derived from: selecting the preserved ganoderma lucidum strain with the passage number less than 3. Wherein the Ganoderma lucidum preserved strain is derived from original Ganoderma lucidum strain obtained by tissue isolation, and the generation number is less than 3 (including 3).
Specifically, the rejuvenation method of the ganoderma lucidum strain special for liquid fermentation comprises the following steps:
(1) shake flask culture screening:
transferring the activated mycelium of the parent strain of the ganoderma lucidum into a shake flask for culture, and screening shake flask fermentation liquor with high mycelium yield and high substrate glucose utilization rate for later use;
wherein, the formula of the shake flask screening is as follows (unit: g/L): 20 parts of glucose, 4 parts of yeast powder, 2 parts of monopotassium phosphate and 2 parts of magnesium sulfate heptahydrate;
(2) plate culture screening:
inoculating fermentation liquor which is obtained by screening in a shake flask and is equivalent to the biological activity index of a parent into a flat plate for culture, and selecting the flat plate with high growth speed for later use;
wherein the plate culture medium is potato glucose agar (PDA culture medium);
the growth rate refers to the time taken for the ganoderma lucidum mycelia to grow over the flat plate;
(3) and (3) shake flask culture verification:
transferring the cultured ganoderma lucidum mycelia on the spare plate into a shake flask for subculture verification, wherein the plate strain used by shake flask fermentation liquor with biological activity indexes equivalent to those of parents and stable passage is rejuvenated ganoderma lucidum strain;
wherein, the formula of the shake flask screening is as follows (unit: g/L): 20 parts of glucose, 4 parts of yeast powder, 2 parts of monopotassium phosphate and 2 parts of magnesium sulfate heptahydrate;
the passage stability index means that the fluctuation range of the mycelium yield and the utilization rate of substrate glucose is within 10 percent after each passage;
wherein the biological activity indexes in the steps (1), (2) and (3) comprise the yield of mycelium and the utilization rate of substrate glucose.
The rejuvenation method of the ganoderma lucidum strain special for liquid fermentation has the advantages that the ganoderma lucidum strain rejuvenated by the method has stable mycelium yield and substrate glucose utilization rate, is equivalent to that of a parent, and has a continuous passage 10 times fluctuation range of less than 10%.
The invention has the beneficial effects that:
the rejuvenation method of the ganoderma lucidum strain special for liquid fermentation can effectively solve the problems of strain degeneration, weak strain activity and poor strain quality caused in the passage process of liquid culture of the ganoderma lucidum strain, improves the mycelium yield, the substrate utilization rate and the product yield of the liquid fermentation of the ganoderma lucidum strain, reduces the pollution rate, and provides strain guarantee for the liquid large-scale sustainable fermentation production of the ganoderma lucidum strain.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
A rejuvenation method of a ganoderma lucidum strain special for liquid fermentation comprises the following steps:
(1) shake flask culture screening:
transferring the activated mycelium of the parent strain of the ganoderma lucidum into a shake flask for culture, and screening shake flask fermentation liquor with high mycelium yield and high substrate glucose utilization rate for later use;
wherein, the formula of the shake flask screening is as follows (unit: g/L): 20 parts of glucose, 4 parts of yeast powder, 2 parts of monopotassium phosphate and 2 parts of magnesium sulfate heptahydrate;
(2) plate culture screening:
inoculating fermentation liquor which is obtained by screening in a shake flask and is equivalent to the biological activity index of a parent into a flat plate for culture, and selecting the flat plate with high growth speed for later use;
wherein the plate culture medium is potato glucose agar (PDA culture medium);
the growth rate refers to the time taken for the ganoderma lucidum mycelia to grow over the flat plate;
the biological activity indexes comprise mycelium dry weight yield and stable utilization rate of substrate glucose;
(3) and (3) shake flask culture verification:
inoculating the cultured Ganoderma mycelia on the spare plate into shake flask for subculture verification, wherein the plate strain used by shake flask fermentation liquid with bioactivity index (mycelium dry weight yield and substrate glucose utilization rate) equivalent to parent strain and stable passage is rejuvenated Ganoderma strain;
wherein, the formula of the shake flask screening is as follows (unit: g/L): 20 parts of glucose, 4 parts of yeast powder, 2 parts of monopotassium phosphate and 2 parts of magnesium sulfate heptahydrate;
the passage stability index means that the fluctuation range of the mycelium yield and the utilization rate of substrate glucose is within 10 percent.
Example 1
Parent ganoderma lucidum strain: ganoderma lucidum G0119 (provided and deposited by the institute of edible fungi, national agrology of Shanghai, accession number: Hunong Lingzhi No.1) was published [ Biotechnology and Bioprocess Engineering 2014, Vol.19, No. 4, 727-.
As shown in the results in Table 1, the Ganoderma strain rejuvenated by the method has yield of dry weight of mycelium (15.12 + -0.21) g/L, substrate utilization rate (64.62 + -2.02)%, plate growth rate (6.39 + -0.30) mm/d before rejuvenation, yield of dry weight of mycelium (15.40 + -0.18) g/L, substrate utilization rate (66.11 + -1.60)%, and plate growth rate (6.42 + -0.21) mm/d after rejuvenation. The yield of mycelium and the utilization rate of substrate glucose before and after rejuvenation are stable, and the fluctuation range of continuous passage for 12 times is less than 8 percent.
Example 2
Parent ganoderma lucidum strain: g.lucidum G0017 (provided and deposited by the institute of edible fungi, national academy of agricultural sciences, Shanghai, accession No.: G.lucidum G0017) has been disclosed [ International Journal of Medicinal Mushrooms, Vol.23, No. 3-53], 2021.
As shown in the results in Table 1, the Ganoderma strain rejuvenated by the method has yield of dry weight of mycelium (13.28 + -0.26) g/L, substrate utilization rate (73.33 + -2.21)%, plate growth speed (5.89 + -0.08) mm/d before rejuvenation, yield of dry weight of mycelium (14.02 + -0.24) g/L, substrate utilization rate (78.05 + -2.32)%, and plate growth speed (5.99 + -0.06) mm/d after rejuvenation. The yield of mycelium and the utilization rate of substrate glucose before and after rejuvenation are stable, and the fluctuation range of 14 continuous passages is less than 9 percent.
Example 3
Parent ganoderma lucidum strain: lucidum (supplied and deposited by the institute for edible fungi, academy of agricultural sciences, Shanghai, under accession number longzhi No.2) has been disclosed in [ Proc. edible fungi proceedings, volume 27, vol.2, 84-91 ].
As shown in the results in Table 1, the Ganoderma strain rejuvenated by the method has yield of dry weight of mycelium (12.25 + -0.12) g/L, substrate utilization rate (73.33 + -2.2)%, plate growth speed (5.53 + -0.30) mm/d before rejuvenation, yield of dry weight of mycelium (12.98 + -0.24) g/L, substrate utilization rate (77.65 + -2.38)%, and plate growth speed (5.73 + -0.18) mm/d after rejuvenation. The yield of mycelium and the utilization rate of substrate glucose before and after rejuvenation are stable, and the fluctuation range of continuous passage for 11 times is less than 7 percent.
In examples 1-3, the comparison of the bioactivity indicators before and after rejuvenation of the Ganoderma lucidum strains is shown in Table 1 below:
table 1 comparison of results before and after rejuvenation in examples
Claims (3)
1. A rejuvenation method of ganoderma lucidum strains special for liquid fermentation is characterized by comprising the following steps:
(1) shake flask culture screening:
transferring the activated ganoderma lucidum mycelia of the parent strains into a shake flask for culture, and screening shake flask fermentation liquor with biological activity indexes equivalent to those of the parents for later use;
(2) plate culture screening:
inoculating fermentation liquor which is obtained by screening in a shake flask and is equivalent to the biological activity index of a parent into a flat plate for culture, and selecting the flat plate with high growth speed for later use;
(3) and (3) shake flask culture verification:
transferring the cultured ganoderma lucidum mycelia on the spare plate into a shake flask for subculture verification, wherein the plate strain used by shake flask fermentation liquor with biological activity indexes equivalent to those of parents and stable passage is the rejuvenated ganoderma lucidum strain.
2. The rejuvenation method of ganoderma lucidum strains specially used for liquid fermentation according to claim 1, wherein the formula of the shake flask culture medium in the steps (1) and (3) is as follows: 10-50 parts of glucose, 2-10 parts of yeast powder, 1-8 parts of monopotassium phosphate and 1-8 parts of magnesium sulfate heptahydrate; unit: g/L; the formula of the plate culture medium in the step (2) is as follows: 10-50 parts of glucose, 2-10 parts of yeast powder, 1-8 parts of monopotassium phosphate, 1-8 parts of magnesium sulfate heptahydrate and 10-20 parts of agar; or using potato dextrose agar plates; unit: g/L.
3. The rejuvenation method for ganoderma lucidum strains dedicated for liquid fermentation according to claim 1, wherein the biological activity indicators include mycelium yield and utilization of substrate glucose; the growth speed refers to the time for the ganoderma lucidum mycelia to grow over the flat plate; the passage stability means that the fluctuation range of the mycelium yield and the utilization rate of substrate glucose is within 10 percent.
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| CN115572754A (en) * | 2022-10-09 | 2023-01-06 | 上海市农业科学院 | Method for evaluating activity of edible and medicinal fungus liquid strain |
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| CN106367352A (en) * | 2016-08-25 | 2017-02-01 | 河北省科学院生物研究所 | Methods for purification and rejuvenation and new variety breeding of edible mushroom strain |
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| CN115572754A (en) * | 2022-10-09 | 2023-01-06 | 上海市农业科学院 | Method for evaluating activity of edible and medicinal fungus liquid strain |
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